Metnase is a human protein that may contribute to genome stability through its role in non-homologous end joining (NHEJ). It contains a SET domain that methylates Histone H3 at lysines 4 &36, which are associated with open chromatin. Additionally, there is a nuclease domain, which recognizes branched structures. Recent studies point to NHEJ having a role in replication and the repair of DNA damage due to replication stress. Our lab has shown Metnase interacts with Ligase IV (LiglV), but not XRCC4. XRCC4- independent NHEJ has not been observed to date;however, others hypothesize that such a pathway exists. We hypothesize that Metnase, along with LiglV, functions in XRCC4-independent NHEJ. The goals of the proposed research are to understand the function and regulation of Metnase in the repair of DNA double strand breaks (DSB) via NHEJ. Additionally, we will investigate how the different domains within the Metnase protein function to regulate protein-protein interactions, and cellular localization of Metnase in response to DNA damage and replication stress. Two aims are proposed to answer these questions: Aim 1: Characterize the recruitment of Metnase to DSBs after DNA damage caused by ionizing radiation or replication stress. Aim 2: Determine the role of Metnase SET/nuclease domains in replication-associated DNA repair. To achieve these goals we will use a combination of molecular and cell biological approaches. Relevance: The completion of these aims will greatly improve our understanding of a critical DNA repair mechanim and will shed light on the role of DNA repair in DNA replication, which is critical for accurate transmission of genetic information from cell-to-cell and across generations to prevent cancer and birth defects. The proposed projects may also reveal new strategies for improving gene targeting, which could increase the efficiency, and reduce the risk, of human gene therapy.